Effect Of LCMR1 On Autophagy In Lung Adenocarinoma Cell Lines

Objective:Lung cancer metastasis related protein 1(LCMRl)was a protein isolated from lung cancer cells with high metastatic potential and was closely related to the occurrence and metastasis of lung cancer.However,its specific mechanism involved in the development of lung cancer had not yet been elucidated.Autophagy,a highly conserved process of cell metabolism,could help tumor cells survive in unfavorable conditions such as starvation.It coud also promote the occurrence and development of tumors.The aim of this study was to find out whether autophagy was one of the mechanisms by which LCMR1 participates in the development of lung cancer.Methods:(1)Lung cancer cell line A549 and H1299 was transfected with LCMR1 shRNA using lentiviral vector.(2)The effects of LCMR1 on cell proliferation were examined by CCK-8 assay,colony formation assay,and cell cycle assay.The effect of LCMR1 on cell migration was detected by wound healing test.The effect of LCMR1 on apoptosis was examined by flow cytometry.(3)To evaluate the effects of LCMR1 on autophagy in lung cancer cells,RT-qPCR and Western blot were used to detect mRNA and protein expressions of autophagy indicator LC3,SQSTM1/p62 and autophagy-related proteins ATG5,ATG13 and BECN1.(4)Western blot was used to detect the expression of PI3K-mTOR pathway and p53.Result:(1)Stable lentiviral vector transfection of LCMR1 shRNA reduced the mRNA and protein expression of LCMR1 in both A549 cells and H1299 cells.(2)Compared with the negative control group and uninfected group,LCMR1 knockdown group in both A549 cell line and H1299 cell line had a lagged behind cell curve,a decreased colony formation rate,an increased cell proportion in the GO/G1 phase and a decreased cell proportion in the S phase of the cell cycle.LCMR1 knockdown had no significant effect on the cell migration rate and the proportion of apoptotic cells in both A549 cells and H1299 cells.(3)The mRNA and protein expression levels of ATG5,ATG13,LC3 and SQSTMl/p62 in the A549 KD group were significantly lower than those in the A549 NC group and A549 group while that the protein expression level of BECN1 was of no difference.In H1299 cells,the mRNA expression levels of LC3,SQSTM1/p62,ATG5,ATG13 and BECN1 in H1299 KD group were lower than that of the H1299 NC group.At the protein level,only the expression of SQSTM1/p62 in H1299 KD group was lower than that of H1299 NC group.There was no statistical difference of ATG5,ATG13,BECN1 and LC3II/LC3I protein expression between H1299 group,H1299 NC group and H1299 KD group.(4)In A549 cell line,the total protein expression of class IPI3K and the ratio of phosphorylated mTOR expression to total mTOR expression were lower in the A549 KD group than those in the A549 NC group and A549 group,while those in H1299 groups were of no significant difference.(5)In A549 cell line,the mRNA expression level and cell nucleus protein expression level of p53 were lower in the A549 KD group than those in the A549 NC group and A549 groupConclusion:(1)LCMR1 knockdown A549 cell line and LCMR1 knockdown H1299 cell line could be successfully constructed through stable lentivirus vector transfection.(2)LCMR1 knockdown inhibited the population growth ability and monomer proliferation ability in both A549 cells and H1299 cells.The increasing cell proportion in G0/G1 phase and the decreasing cell proportion in S phase,indicating that LCMR1 knockdown A549 cells and H1299 cells were in GO/G1 phase arrest.(3)LCMR1 knockdown did not affect the migration ability and apoptosis of both A549 cells and H1299 cells.(4)In A549 cells,LCMR1 knockdown significantly inhibited both mRNA and protein expression of ATG5,ATG13 and SQSTM1/p62,decreased the ratio of LC3II/LC3I,and decreased the basal level of autophagy.It was speculated that LCMR1 inhibited the initial phase of autophagy in A549 cells without affecting the binding of autophagosomes to lysosomes.(5)LCMR1 knockdown did not affect the level of autophagy and expression of ATG5,ATG13 and BECN1 in H1299 cells.(6)The low level of autophagy caused by LCMR1 knockdown could feedback to inhibit the activity of PI3K-mTOR pathway.(7)LCMR1 knockdown reduced protein expression level of p53 in cell nucleus.P53-dependent autophagy may be one of the mechanisms by which LCMR1 participates in the development and metastasis of lung adenocarcinoma.